- Authors: Boix-Besora A. et al.
- Year: 2022
- Journal: Vaccines 10 250
- Applications: in vitro / DNA / PEIpro
- Cell type: HEK293SF-3F6
Description: Human embryonic kidney Fibroblast cell line genetically modified with a truncated version of EBNA1 which grows in suspension and chemically defined serum-free medium.
Exponentially growing HEK293 cells were passaged in order to have a cell density of 2·106 cells/mL at transfection time. PEIpro (Polyplus-transfection) was used as a transfection reagent. PEI-DNA complexes were formed under sterile conditions, by adding PEI to a plasmid DNA mixture diluted for a total DNA concentration of 1 µg/mL in fresh culture media (10% of the total volume of cell culture to be transfected). The mixture was incubated for 15 min at RT and then added to cell culture. The ratio between plasmids and transfection reagent was optimized using a Box–Behnken design of experiments and described in the next section.
Virus-like particles (VLPs) constitute a promising approach to recombinant vaccine development. They are robust, safe, versatile and highly immunogenic supra-molecular structures that closely mimic the native conformation of viruses without carrying their genetic material. HIV-1 Gag VLPs share similar characteristics with wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, making them a suitable platform for the expression of its spike membrane protein to generate a potential vaccine candidate for COVID-19. This work proposes a methodology for the generation of SARS-CoV-2 VLPs by their co-expression with HIV-1 Gag protein. We achieved VLP functionalization with coronavirus spike protein, optimized its expression using a design of experiments (DoE). We also performed the bioprocess at a bioreactor scale followed by a scalable downstream purification process consisting of two clarifications, an ion exchange and size-exclusion chromatography. The whole production process is conceived to enhance its transferability at current good manufacturing practice (cGMP) industrial scale manufacturing. Moreover, the approach proposed could be expanded to produce additional Gag-based VLPs against different diseases or COVID-19 variants.